Project Summary/Abstract: Cyclic dinucleotides (CDNs) play important second messenger roles in Gram-negative bacteria, regulating a number of different types of bacterial processes such as motility, biofilm formation, host colonization, bacterial growth, and metabolism. CDNs levels are tightly controlled by bacterial encoded diguanylate/diadenylate cyclases and phosphodiesterases. In addition to regulating bacterial life cycle processes, CDNs can alert the innate immune response during infection. A major cytosolic sensor for intracellular CDNs in animal hosts is STING, which upon its activation, triggers an innate immune response through the induction of type I interferon and pro-inflammatory cytokines. As such, synthetic CDNs have been applied exogenously prior to infection to stimulate inflammasome activity and reduce the load of Chlamydia trachomatis, a Gram-negative, obligate intracellular bacterium. Thus CDNs can be used to initiate an immune response to therapeutically treat Chlamydia infection, in addition to standard two-week antibiotic therapy. Another Gram-negative, obligate intracellular, macrophage-tropic bacterium is Coxiella burnetii, which is the causative agent of the zoonotic disease Q fever. The primary route of Coxiella transmission is through aerosols. However, Coxiella is also found in ticks, and they have been implicated as vectors. Acute phase of the disease in humans is characterized primarily by influenza-like symptoms, and individuals that develop chronic infection must undergo 18-24 months of antibiotic therapy. We contend that alternative methods to current antibiotic treatments need to be developed to reduce Coxiella load in infected animals. Our preliminary data suggests that CDNs are produced during Coxiella infection and that the Coxiella genome contains putative genes that encode diguanylate/diadenylate cyclases and phosphodiesterases. However, little is known about how CDNs elicit a STING-mediated host response during Coxiella infection. Thus, we propose to determine how CDNs control the innate immune response to Coxiella burnetii infection. In Aim 1, we will characterize the STING-mediated host response to Coxiella infection in vertebrate and invertebrate models. In Aim 2, we will dissect STING's mechanism of action during Coxiella infection with regard to caspase activation and programmed cell death. In Aim 3, we will stimulate the host innate immune response with CDNs in vertebrate and invertebrate models to reduce the magnitude of Coxiella infection. Together, the proposed work will characterize the CDN-mediated innate immune response to Coxiella infection and how we can exploit CDNs to reduce overall Coxiella burden. The use of invertebrate models of Coxiella infection may be applicable to other vector-borne diseases. The information gained in this study will have broad-ranging impacts in innate immunity towards to the development of new therapies to treat Coxiella infection, and we will identify potentially novel CDN/STING-mediated mechanisms of immunity that will be applicable to other pathogenic infections.